We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress  and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H Pseudomonas aeruginosa (PA) is an important human pathogen that is currently ranked fifth by the World Health Organization in overall infections . After the addition of purified OxyR (500 and 1000 nM), the reaction was incubated at 25°C for 15 min. Subsequently, 25 μ l of 0.2 mM Mg2+-0.1 mM Ca2+-0.5 U of DNase I was added to the binding reaction, and incubated for an additional 1 min. Finally, 200 μl of stop solution  was added. The mixture was then extracted with phenol-chloroform and the DNA precipitated with 95% ethanol at -20°C. The pellets were resuspended in sequencing buffer and loaded onto a 5% denaturing polyacrylamide sequencing gel. After transfer of the sequencing gel to PVDF membranes, the OxyR-specific footprint was assessed by autoradiography.A radioactively labeled DNA fragment harboring putative OxyR recognition sequences was prepared by PCR. The reaction was initiated using a PA1310FW primer  impregnated with 10 μl of either 0.3 M or 0.5 M t-BOOH were placed on the top-agar surface and the plates were incubated at 37°C for 24 hr. The zones of killing were measured and the results are presented as the average from at least 3 independent experiments +/- standard error (SE).An exponential phase bacterial suspension was added to 5 ml of fresh L-broth containing 0.8% low-melting-point agarose (SeaPlaque) to a final O.D.PA and selected isogenic mutants to degrade t-BOOH was investigated as previously described [PA bacteria  were inoculated into L-broth to a final O.D.600 of 0.1 and allowed to grow at 37°C with shaking for ~3 hr, at which point various cells were diluted into fresh L-broth at an O.D.600 of 0.4. Then, 200 μM of t-BOOH was added to each culture. The remaining amount of t-BOOH was determined at 0, 6, 9 and 12 min, respectively. At each time point, 1 ml of untreated and treated cells were collected by centrifugation to clarify the bacteria. An aliquot (100 μl) was added to 400 μl of 25 mM sulfuric acid followed by 500 μl of freshly prepared reaction buffer . The reaction was incubated for 10 min at room temperature and the optical density of the solution measured at 540 nm. The concentration of remaining t-BOOH in the culture was calculated using a standard curve that was repeated for each set of experiments. To investigate the potential ability of purified PhnW to degrade or modify t-BOOH, PhnW (see below for purification details) was added to a solution containing 200 μM of t-BOOH and 100 μM of the reductant, dithiothreitol (DTT). Finally, the reaction mixture was incubated at 37°C and investigated for the degradation of t-BOOH as mentioned above at 15, 30 and 60 min, respectively.The ability of escribed . BrieflyphnW coding region was amplified from PA genomic DNA using Pfu DNA polymerase with primer phnW F-pET BamHI (5-ACTGGATCCATGAGCACTGCCGAACGCGCACCCAT-3´) and primer phnW R-pET NotI(5´-TATGCGGCCGCGATCTCGAGGACTTCCAGTTCGCGCAGC-3´). The expected PCR product which is ~1.1 kb was digested with BamHI and NotI and ligated within compatible sites of the expression vector, pET23a, creating pET-phnW. E. coli BL21(DE3)/pLysE containing pET-phnW was grown in L-broth supplemented with 100 μg/ml ampicillin at 37°C with shaking until the O.D.600 of the cells reached 0.6–0.8. Then, 1 mM IPTG was added and incubated for 3 hr to induce the expression of PhnW. The cell pellet was kept at -20°C until use. To initiate purification of this protein, the cell pellets were resuspended in sonication buffer . After sonication with a Sonic Dismembrator Model 500 for 15 min on ice, the supernatant was separated from cell debris by centrifugation at 13,000 x g for 30 min at 4°C. The supernatant was loaded on a Ni-NTA agarose column pre-equilibrated with sonication buffer. Non-specific binding proteins were removed with 10 column volumes (CVs) of washing buffer (sonication buffer with increasing imidazole concentration to 50 mM). PhnW was eluted from the Ni-NTA agarose column using 5 CVs of elution buffer (sonication buffer with increasing the imidazole concentrations to 100 mM). The fractions containing the PhnW protein were pooled and dialyzed against 50 mM Tris, pH 8.0, 5% glycerol, 0.05 M NaCl and 0.1 mM EDTA.The http://www.genome.jp/kegg/pathway.html) was used to identify where PhnW lies in fundamental PA metabolism. First, after clicking on the “Organism” bar, we typed in “Pseudomonas” and scrolled down to PA strain PAO1 (codename “pae”). We then scrolled down to section 1.6 (Metabolism of other amino acids) and selected subsection 00440 for “phosphonate and phosphinate metabolism.” The phnW gene is coined PA1310 in the PA PAO1 genome (www.pseudomonas.com) and we subsequently entered phnW in the open box and hit return. An elegant pathway map is given with the PhnW enzymatic reaction presented in red font with the enzyme commission number of 2.6.1.37 in S2 Fig.The KEGG Pathway Database  and PA3n locus, . We werealases A  and B [6 et. al.  who idenoxyR mutant and wild-type bacteria. Our results revealed that 3/40 genes could not be amplified (even with 1 μg of RNA). Duplicate results of the RT-PCR analysis revealed differences in gene expression of 15 genes compared between wild-type bacteria and the oxyR mutant. These included mvfR, qscR, PA2957, PA0846, betX, PA0192, PA1097, PA1423, phnW, PA1648, PA3242, PA4403, PA2999, PA0594 and PA5095 (Table 2). Among this group, the RT-PCR results of the phnW gene, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, revealed lower gene expression levels in the oxyR mutant when compared to wild-type bacteria under non-inducing conditions , which was also confirmed transcriptionally by ß-galactosidase activity assays . The upstream sequence that is considered a putative OxyR binding domain of phnW matched 12 of 16 bases. Two sets of control genes were used; the omlA gene which is a constitutively expressed internal control [ahpC, which is a known member of the PA OxyR regulon [Our first experiment to corroborate our bioinformatic predictions that OxyR either positively or negatively controls transcription of specific genes was by performing RT-PCR from exponential phase bacteria. Genes that matched 9–13 of 16 nucleotides of the OxyR binding motif used in our bioinformatic search that did not encode predicted hypothetical proteins , were amplified and compared between an phnW, bdlA, bexT and qscR) that showed lower expression in the oxyR mutant were further evaluated by transcriptional ß-galactosidase reporter assays. Herein, we focused only on the phnW gene. The upstream region of the phnW gene which contains the OxyR binding domain was cloned into pQF50, a transcriptional fusion vector [oxyR, oxyR-attB oxyR, oxyR-attB oxyR C199S, as well as ahpC, ohr or ahpC ohr double mutant bacteria. As a reminder, AhpCF and Ohr in PA are two of the major proteins involved in the cytoplasmic detoxification of organic hydroperoxides [phnW gene expression is lower in the oxyR mutant than in wild-type bacteria . Our results also showed complementation of the phnW mutant in both cell backgrounds harboring either OxyR or OxyR C199S (constitutively OxyR reduced form), the latter of which is unable to sense oxidative stress [phnW was also ~30% lower in the ahpC mutant while it was 80% lower in the oxyR mutant when compared to wild-type levels. Still, phnW expression could be induced to approximately parental levels by exposure to 250 μM of t-BOOH, but not in ohr or ahpC ohr mutant bacteria . However, exposure of ahpC ohr mutant bacteria to 250 μM t-BOOH caused a slight reduction (less than 10%) in phnW expression .From our previous experiments using RT-PCR, only 4/15 genes . To investigate the specific binding of OxyR on the putative OxyR-binding site upstream of phnW, the unrelated protein (BSA) or unrelated DNA (pUCP20) was added to the reaction. Our results showed no change in the mobility shift between the phnW fragment and OxyR protein (see UP and UD inrespectively). These data revealed the specific binding of OxyR on the putative OxyR binding sequence upstream of the phnW gene.Next, we assessed the binding of purified OxyR to a 199-bp DNA fragment upstream of the phnW promoter that was based upon our bioinformatic search using a DNase I footprinting analysis. Various concentrations of purified OxyR protein were used. The footprint pattern showed that OxyR at 500 nM binds to these upstream sequences . This putative OxyR binding domain is shown in bold, italic, and lower case nucleotide sequences in Fig 4B. We also performed a computer-based promoter prediction derived from 500-bp upstream of the phnW gene using the BDGP: Neural Network Promoter Prediction program  and discovered a weak promoter score of 0.49; a perfect promoter score for this particular program is 1.0 (http://www.fruitfly.org/seq_tools/promoter.html). Such a low score indicated that the phnW gene is not a prioritized gene from the overall regulatory scheme in PA.We next determined the precise location of the OxyR binding domain within the t-BOOH of the isogenic phnW mutant relative to a number of mutants, complemented and wild-type strains was next examined. Our preliminary results indicated that the phnW mutant is only susceptibility to t-BOOH, and not to CHP and H2O2, respectively. Our results showed that the phnW mutant  was more susceptible to t-BOOH compared to wild-type and complemented bacteria  in the exponential but not the stationary growth phase . In contrast, using stationary phase organisms, the oxyR phnW mutant was slightly more sensitive to t-BOOH than the oxyR mutant , while provision of the phnW gene to the oxyR mutant provided some measure of protection against t-BOOH . Moreover, provision of the phnW gene in trans could also protect a strain lacking AhpC and Ohr proteins (ohr ahpC mutant), that have previously been shown to be highly susceptible to organic hydroperoxides including t-BOOH [ohr ahpC double mutant from t-BOOH killing  degradation by exponential phase, aerobic bacteria was measured in selected bacteria and the results were expressed as the percent of t-BOOH remaining in the media after 12 min incubation (. Wild-type bacteria (white bar) rapidly degraded t-BOOH, as did an oxyR mutant containing pUCP-oxyR (white bar with dots) or a phnW mutant containing pUCP-phnW (darkgray bar). As expected, the oxyR mutant revealed the slowest t-BOOH degradation rate (light gray bar) compared with all test strains. At t-BOOH exposure times from 0 to 9 min, the phnW mutant showed no difference in the rate of t-BOOH degradation compared to that of oxyR/poxyR, phnW::Gm/pphnW and wild-type bacteria, but clear differences were evident in t-BOOH degradation at about 12 min after exposure (. The phnW mutant degraded only 55% of the t-BOOH compared to the 80% degradation observed in wild-type bacteria at the end of assay (12 min). The ability of purified PhnW to degrade t-BOOH also investigated. However, Purified PhnW did not show a direct ability to degrade t-BOOH in vitro.To determine whether PhnW influences the degradation rate of employed . The ratPA and other Gram-negative bacteria including E. coli, S. typhimurium and PA to H2O2 and alkyl hydroperoxides is governed by the major LysR family member and trans-activator, OxyR [2O2 or organic hydroperoxides [S-nitrosylation in E. coli, events that are distinct from OxyR-mediated gene activation under oxidative stess conditions [PA OxyR and various antioxidants under its control for more than 17 years [PA OxyR-dependent genes that would not be predicted to play a classical role in the oxidative stress response. First, we used the DNA motif ATAG-N7-CTAT-N7-ATAG-N7-CTAT that is the E. coli OxyR consensus binding sequence to search the PA genome database for genes that are possibly regulated by PA OxyR. When compared to a previous study by Wei et al., [2O2, we found that only 8 of the 41 genes listed in Table 2 matched those identified in this study. These genes were betX, ahpB, ahpC, bdlA, katB, nadA, mscL and mvfR, respectively. Among this gene list, phnW was not found by Wei et al., [phnW gene [The major response of or, OxyR ,20,21,22eroxides . Still, nditions . Howevernditions . We have17 years ,6,17,22. et al., , who idehnW gene .phnW gene, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, can be found in the genomes of both pathogenic and non-pathogenic bacteria [phn-based genes are all down-regulated  when PA was grown in blood from burn patients, a phenomenon that is not understood at the mechanistic level [PA PhnW catalyzes the transfer of the amino group of 2-aminoethylphophonate (ciliatine) to pyruvate, resulting in the production of 2-phosphonoacetaldehyde and alanine [phnW gene was constitutively expressed, independent of whether OxyR was in its reduced or oxidized states, yet its expression was reduced 80% in an oxyR mutant. In contrast, phnW expression was found to be induced 30% in ahpC mutants relative to wild type bacteria . The significantly lower phnW expression in the oxyR mutant indicates that OxyR is a regulator of the phnW gene . This was confirmed by the ability of OxyR to bind specifically to phnW upstream sequences using both EMSA and DNase I footprinting analyses Figs  and 3.PA OxyR contains the four putative OxyR binding tetranucleotide sequences with 67% identity to similar sequences in E. coli [et al. showed that as little as 84 nM OxyR can bind to the promoter region upstream of the ahpC gene [phnW gene. This result was also confirmed by our DNase I footprinting results, indicating that very high concentration of OxyR (500 nM) are needed for binding. A previous study by Ochsner et. al. [katB, ahpB or ahpC. In addition, DNase I footprinting of E. coli OxyR on the ahpC promoter was studied by Zheng et. al. [phnW upstream nucleotides compared to the major OxyR-dependent antioxidant gene, ahpC [phnW expression in PA. Moreover, it is also supported by the alignment of the putative OxyR binding sequence of phnW with other OxyR-dependent genes base on the results of Wei et al., [phnW is 80% identical to betX (PA3236), 53% to the ahpB, katB and ahpC genes and 46% to bdlA, respectively , while an absence of both AhpCF and Ohr allow for wild-type phnW expression. This may be a compensatory strategy triggered by PhnW for organisms to protect themselves from t-BOOH toxicity.The et. al., . Our rest-BOOH in vivo, we sought in earnest to mechanistically define how this might occur. The forward substrate of PhnW, 2-aminoethylphosphonate, is taken into the cell by an ABC transporter system and can be utilized as a sole carbon source for growth [t-BOOH degrading activity and we subsequently proved this experimentally. Rather, we posited that L-alanine, one product of PhnW, could modulate cellular antioxidant levels. L-alanine has been shown to trigger activation of an antioxidant response in eukaryotes [2 molecule per pyruvate molecule. The significance of an increase in reducing power (electrons) is that many alkylhydroperoxide reductases such as the E. coli AhpCF require NADH as a bound cofactor [see panel B inS2 Fig, black arrows on upper left) which can be funneled into the glycolytic pathway, leading to more reducing power starting with the two NADH molecules produced from pyruvate dehydrogenase and the following six NADH and 2 FADH2 molecules from the TCA cycle. The substrate of PhnW protein (aminoethylphosphonic acid) was also investigated its ability to help protect bacteria from t-BOOH toxicity. Our results demonstrated that supplementation of aminoethylphosphonic acid in the media slightly helped to protect bacteria from t-BOOH toxicity at concentrations as low as 1 mM or as high as 10 mM. Surprisingly, the protection afforded by bacteria grown in the presence of aminoethylphosphonic acid was also specific to the ahpC ohr double mutant only. The killing zone with 0.2 M t-BOOH of stationary phase an ahpC ohr double mutant was 42±0.063 mm and 40±0.031 mm for either control bacteria or organisms supplemented with 10 mM aminoethylphosphonic acid, respectively.Because PhnW offered protection against r growth . Still, karyotes . Alternacofactor . Anothert-BOOH resistance not directly but possibly indirectly, we propose the following model of phnW expression under different cellular conditions that help protect bacteria from t-BOOH. A cartoon depicting various oxidative conditions in selected strains is shown in Fig 7A–7D. First, there is continuous expression of phnW in wild-type bacteria .Given the frustrating caveat of not knowing the true mechanism by which PhnW influences t-BOOH. We expose a dynamic that involves a complex interplay between PhnW, that does not appear to have the direct capacity to degrade t-BOOH, but an absence of it in the bacteria shifts the overall expression of other classical AHPs including AhpCF and Ohr. Further studies are required to elucidate the precise detoxification mechanism that PhnW provides to aerobically grown PA.In this study, we revealed a previously unrecognized gene under the control of PA OxyR that plays an as yet unknown role in detoxification of the organic hydroperoxide, S1 FigphnW. The MView software from EMBL-EBI was used. The conservation of these sequences in OxyR-dependent genes such as phnW, betX, ahpB, katB, ahpC and bdlA are shown in black. Dark gray represent the nucleotides homologous to the OxyR putative binding region of phnW while lighter gray are not.The OxyR putative binding region of several OxyR-dependent genes based on (18) was used to compare to OxyR binding region of (TIFF)Click here for additional data file.S2 FigA. Gene orientation of the phnW gene on the PA PAO1 genome. B. KEGG map of a portion of central metabolism of PA with the PhnW protein catalyzing the reaction shown as an arrow. The black arrows on the upper left corner of the diagram indicate the potential flow of acetate and acetaldehyde back in the glycolytic pathway.(TIFF)Click here for additional data file.


Enterobacteriaceae (ESBL-E). To prevent the overuse of broad-spectrum antimicrobial therapy, the main objective of this study was to test the performance of digestive colonization surveillance as a predictor of ESBL-E presence or absence in respiratory samples performed in ICU and to evaluate the impact of time sampling (≤5 days or >5 days) on such prediction. Design: Multicentric retrospective observational study, including every patient with a respiratory tract specimen positive culture and a previous rectal ESBL-E screening performed within 7 days before the respiratory sample, between January 2012 and December 2014. Results were analyzed in two groups: respiratory samples obtained during the first 5 days of ICU stay (early group) and respiratory samples obtained after 5 days (late group). Interventions: none.The choice of empirical antimicrobial therapy for pneumonia in intensive care unit (ICU) is a challenge, since pneumonia is often related to multidrug-resistant pathogens, particularly extended-spectrum β-lactamase-producing Among 2498 respiratory tract samples analyzed corresponding to 1503 patients, 1557 (62.3%) were performed early (≤5 days) and 941 (37.7%) later (>5 days). Positivity rates for ESBL-E were 15.0 and 36.8% for rectal swabs in the early and late groups, respectively. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values and likelihood ratios were calculated for ESBL-E digestive colonization as a predictor of ESBL-E presence in respiratory samples. PPVs of ESBL-E digestive colonization were 14.5%  and 34.4% , for the early and late groups, respectively, whereas NPVs were 99.2%  and 93.4% , respectively.Systematic surveillance of ESBL-E digestive colonization may be useful to limit the use of carbapenems when pneumonia is suspected in ICU. When rectal swabs are negative, the risk of having ESBL-E in respiratory samples is very low even after 5 days of ICU stay.The online version of this article (doi:10.1186/s13613-017-0237-x) contains supplementary material, which is available to authorized users. Enterobacteriaceae (ESBL-E) ) and late (93.4% [91.9–95.0]) groups were due to the low prevalence of ESBL-E in respiratory samples. However, in this study, the overall prevalence of ESBL-E in early and late respiratory samples was 8.1% (203/2498), which was higher than the prevalence described in the French ICU nosocomial infection surveillance network in 2013 (6.4%) and 2014 (4.9%) , 27. ConThe link between rectal swabs and respiratory samples positive for EBSL-E is based on a debatable hypothesis, suggesting a contamination from digestive flora to the respiratory tract . The evoEnterobacteriaceae species. Indeed, the association of ESBL-E presence or absence in rectal swab and respiratory sample is relevant for the choice of empirical antimicrobial therapy, whatever the Enterobacteriaceae species. In addition, ESBL-E are transmitted through plasmids from an Enterobacteriaceae strain to another, making the concordance interpretation between respiratory and rectal ESBL-E difficult.However, there are some limitations to our study. As high as 27% of the culture-positive samples have been excluded due to the absence of rectal swab. Moreover, the weight of patients who had multiple respiratory sampling may have influenced the results. The evidence of a clinical benefit of ESBL-E digestive colonization surveillance to predict the presence or the absence of ESBL-E in respiratory samples needs to be confirmed in a clinical study involving pneumonia and not only respiratory samples. As we chose to focus on respiratory samples and not episodes of pneumonia, we did not study antimicrobial therapy regimens. We chose to focus on ESBL-E presence or absence in respiratory samples and in previous rectal swabs, regardless of the Systematic surveillance of ESBL-E digestive colonization may be useful to limit the use of carbapenems when pneumonia is suspected, particularly in the late phase of ICU stay. When the rectal swab is negative for ESBL-E, whatever the length of stay in ICU, carbapenems may not be used. The evidence of clinical benefit of ESBL-E digestive colonization surveillance to predict the presence or the absence of ESBL-E in respiratory samples needs to be confirmed in a large prospective clinical study.


Tetranychus cinnabarinus, an important pest in agriculture. The gamma aminobutyric acid (GABA) contents in a laboratory abamectin resistant strain of T. cinnabarinus (AbR) were significantly increased. Decreases in activity and mRNA expression of GABA transaminase (GABA-T) were responsible for GABA accumulation in AbR mites. To clarify the mechanism of GABA accumulation mediated abamectin resistance, three artificial approaches were conducted to increase GABA contents in susceptible mites, including feeding of vigabatrin (a specific inhibitor of GABA-T), feeding of exogenous GABA, and inhibition of GABA-T gene expression. The results showed that susceptible mites developed resistance to abamectin when the GABA contents were artificially increased. We also observed that the mites with higher GABA contents moved more slowly, which is consistent with the fact that GABA is an inhibitory neurotransmitter in arthropods. Subsequently, functional response assays revealed that predation rates of predatory mites on GABA accumulated abamectin-resistant mites were much higher than control groups. The tolerance to abamectin, slow crawling speed, and vulnerability to predators were all resulted from GABA accumulation. This relationship between GABA and predation was also confirmed in a field-collected population. Our finding indicates that predatory mites might be used as a tool for biological control to circumvent the development of abamectin resistance in mites.Abamectin has been widely used as an insecticide/acaricide for more than 30 years because of its superior bioactivity. Recently, an interesting phenomenon was identified in the carmine spider mite,  Spider mites have been one of the most polyphagous arthropods on this planet. They feed on more than 1,100 different plants in the field or in green houses  (Acarina: Tetranychidae), is widely distributed around the world. For many years, the control of T. cinnabarinus has traditionally relied on sprays of acaricides and control has been reported , GABA, widely present in most prokaryotic and eukaryotic organisms  was collected from the fields of Beibei District, Chongqing, China in 1998, and was transferred to fresh cowpea seedlings without pesticide treatments. The abamectin resistant strain (AbR) was continuously selected from SS with abamectin in the laboratory and has about 30-folds of resistance level compared with the SS strain. The fenpropathrin resistant strain (FeR) was also selected from SS with fenpropathrin continuously and has about 100-folds of resistance level. The FeR was used as a control resistant strain in this study. Wild strain (WS), a field population with 2.5-fold of resistance level, was collected from the fields of Beibei, Chongqing, in 2015.The rearing conditions of all strains were: 26 ± 1°C temperature, 35–55% humidity, and 14:10 (L: D) photoperiod.The leaf disk method was used in chemical treatment. Water was added to a glass culture dish with a diameter of 9 cm, and a sponge (3 × 3 × 2 cm) was placed in each dish. The sponge was wetted by water and covered by a piece of filter paper. Smooth cowpea leaves were put on the wet filter paper with their back facing up. Three-day old healthy adult females were gently transferred to the leaves and placed for 2 h to make them stable. Potter spray tower  was used to spray desired concentrations of abamectin, GABA or vigabatrin on the SS, and the treated mites were recorded as SS-A, SS-G and SS-V, respectively.Toxicity bioassays were measured with modified residual coated vial (RCV) method  from different strains and different treatments were placed into the middle of a glass rod (10 cm long and 0.5 mm diameter). The time that the mite crawled from the mid-point to either end of the rod continuously and straightly was recorded to calculate the crawling speed. One hundred mites were measured in each strain or treatment group.Neoseiulus barkeri (Hughes), was placed in the middle. When it crawled across the water to certain leaf was regarded as a choice. Three replicates with at least 30 predatory mites were conducted.The selection frequencies of predatory mites were measured by a four-side device. The cross style sponge, covered by filter paper in the same size, was placed into 6cm-diameter-dish and soaked in water. A circular black light paper with 2 cm diameter was placed in the central part and four circular leaves with 2 cm diameter were placed in four directions 2 mm apart from the center. Mites from different strains or treatments were placed on three directions of the device, respectively, and the remaining direction was set as blank control. A 24-h starved predatory mite, According to McMurtry and Scriven , the spoThe chromatographic conditions used for this study were as follows: A Hitachi L-2000 HPLC system (UV detector) and an Alltima C18 reverse phase column were utilized. Column temperature was 40°C with UV at wave length of 254 nm. Mobile phase A: acetonitrile (70 mmol/L)/acetic acid buffer (pH 6.5) = 25/975 (v/v); mobile phase B: acetonitrile/water/methanol = 450/400/150 (v/v); washing conditions A/B = 50/50 (v/v); flow rate was 0.8 mL/min and washing time was 15 min. Standard GABA samples and sample extracts (100 μL) were injected into test tubes. Drying agents (methanol/sodium acetate (1.0 mol/L)/triethylamine = 2/2/1 (v/v)) and 50μL of freeze-dried derivatizing agent (4-chlorophenylisothiocyanate/methanol/ethanol/triethylamine/water = 1/6/1/1/1 (v/v)) were also added. The derivatizing reaction occurred at room temperature for 30 min. The solution was freeze-dried and stored at 4°C. The sample was dissolved with 1mL diluting solution , and 10 μL of this sample was used for GABA content assessment. The diluting buffer was used as negative control. The entire experiment was replicated three times with three determinations per sample.Two hundred adult females were homogenized in 1 mL PBS (0.05 mol/L pH7.0) on ice, then centrifuged at 10,000 g for 10 min at 4°C. The supernatant was used for testing. Protein concentration was measured by Coomassie brilliant blue method  were then added to the reaction for 60 min incubation at 30°C. The reaction was measured at 340 nm using microplate reader. The experiments were repeated for three times.To test the activity of GABA-T, γ-aminobutyrate and α-ketoglutarate (1:1) were used as substrates for 15 min incubation at 30°C. Enzyme solution and sodium phosphate .  Pfaffl, . The qPCTC1 and GABA-TTC2 were highly homologous, primers (GABA-T-RNAi) with T7 promoter was designed to amplify a common region of GABA-TTC1 and GABA-TTC2, and dsRNA was synthetized using TranscriptAid T7 High Yield Transcription Kit . Primers (GABA-T-qPCR) used in qPCR were outside the dsRNA region for detection of RNAi efficiency of both GABA-TTC1 and GABA-TTC2. GFP dsRNAs were used as controls. Primer information was presented in Table Since the nucleotide sequences of GABA-TdsRNA-GABA-T to knock down the expression of GABA-T genes in T. cinnabarinus were prepared as follows: cowpea leaves were cut to a 1.5 cm diameter feeding arena, incubated at 60°C for 3 min for dehydration, and then separately treated with DEPC-water, GFP dsRNA, and dsRNA-GABA-T (10 μg/per leaf) for 5 h. After fully absorbed, the leaves were put on wet filter paper. Thirty female adults (3–5 d old and starved for 24 h) were placed in each leaf-disc. After feeding for 48 h, the mites were collected for the subsequent experiments.Leaf-discs containing T. urticae  genes had no significant difference among susceptible (SS), fenpropathrin-resistant  and abamectin-resistant strains (AbR)  Figure . However) Figure .The enzyme activities of GAD and GABA-T, which catalyze the synthesis and catabolism of GABA, respectively, were also compared between SS and AbR. In the adult , GABA-T activity in AbR was significantly lower than that in SS Figure . HoweverT. cinnabarinus, vigabatrin, a specific inhibitor of GABA-T, was used to treat the mites. The susceptible CSMs of SS strain were used to perform experiments since they have not developed GABA related resistance to abamectin. The activity of GABA-T could be inhibited in vivo by vigabatrin (0.443 mM) and 22.76% of activity was inhibited after susceptible CSMs were exposed with vigabatrin for 4 h . However, the GABA returned to almost the same level 8 h after the treatment  also increased about 2-times after sprayed with abamectin (6.25 μM) after 4 h and maintained in the high level after 8 h (SS-A8), suggesting that abamectin could stimulate GABA release in mites for more than 8 h  showed that the GABA level in mites increased about 2-times after treating with vigabatrin (0.443 mM for 4 h) t Figure , SS-V8, h Figure . HoweverT Figure  and the T Figure .30or LC50of abamectin, respectively  treatment, the result of toxicity test showed that the resistance of SS against abamectin increased 4.1-fold Table . The eff mM treatThe measurement of crawling speed of CSMs in different treatments revealed that the AbR mites moved more slowly than SS and FeR (resistant strain control) mites, whereas there was no significant difference between SS and FeR mites Figure . When SSMites from SS, AbR, and FeR strains were placed in three different directions of a four-side device (the rest direction was set as blank control) Figure . Then, adsRNA-GABA-T, which possessed higher GABA contents, was significantly stronger than that to controls (fed with water or dsRNA-GFP)  Table .To clarify if the phenomenon observed in our laboratory strains is also present in randomly collected field strains, a wild strain (WS) was collected to validate the GABA effect. This WS strain has 2.5-fold abamectin resistant ratio compared with the SS strain. Our results showed that there was no significant difference in GABA contents, GABA-T mRNA level and crawling speed between SS and WS strains Figure . HoweverAs the two most important enzymes in the GABAergic neurons, GAD is responsible for synthesis of GABA while GABA-T can metabolize GABA into SSA. In living organisms, either an increase of GAD activity or a decrease of GABA-T activity can result in the accumulation of GABA  to inhibit GABA-T's activity, supplying exogenous GABA to increase GABA contents and suppressing gene expression of GABA-T via RNAi, were adopted to elevate the GABA level artificially. Their effects on increasing GABA contents in mites were confirmed by detecting the amount of GABA with HPLC and testing behavior responses with crawling speed measurements. The results of bioassay revealed that these GABA-elevated mites became less sensitive to abamectin than controls, which leads to the question why the increase of GABA contents can cause abamectin resistance in mites? One mode of action of abamectin was reported as stimulating the release of GABA at the brain synaptosomes in rats  was associated with higher deleterious effects than Ester (locus for two esterase enzymes)  are more correlated with over-expression of detoxifying enzyme genes while fitness costs on behavioral traits  are more correlated with the insensitivity of target sites in the nervous system. Estimations of fitness costs from Plutella xylostella had resulted in significant biological fitness costs  on T. cinnabarinus indicated that the predation rates of N. barkeri on abamectin-resistant mites and susceptible individuals with an artificially increased GABA contents were much higher than that on susceptible mites. Similar to susceptible mites in the laboratory, when GABA-T activity of a field population was inhibited by RNAi, GABA contents in field mites and the predation rates of N. barkeri were higher than those in controls, indicating that the “GABA effect” exists not only in laboratory stains but also in wild population. Theoretically, predator' preference could affect the predation efficiency against different prey. The N. barkeri did show preference on leaf-disc with mites compared with empty control. However, the predatory mites showed no significant preference between acaricide-resistant and susceptible mites, from which the possibility of prey-selectivity preference contributing to higher predation rates can be excluded. Therefore, we conclude that the higher predation rates of N. barkeri on higher GABA-content mites attribute to the fact that these mites moved more slowly than individuals with normal GABA contents. The abamectin resistance in T. cinnabarinus confers a predation cost. The predation cost had also been documented in other pesticide resistant species. For instance, knock-down resistance to insecticides in Myzus persicae imposed a fitness cost through increased vulnerability to natural enemies  in Culex pipiens increased the probability of predation at both the larval and the adult stage  contents caused abamectin resistance in mites. On the other hand, high GABA contents also made mites more vulnerable to their natural enemies.LH, GJS, JN, and QX designed research; ZX, YL, PW, and WL performed research; ZX, YL, PW, GS, LH, and QX analyzed data; ZX, KF, GS, WX, and JN. JW, GJS, QX, and LH wrote the paper.This research was funded in part by the National Natural Science Foundation of China (31672085), the China Postdoctoral Science Foundation (2016M602638), the Fundamental Research Funds for the Central Universities (XDJK2016A005), and the Foundation Project of Southwest University (SWU114049). This work was also supported by Abilene Christian University Faculty Renewal Leave Grants, Pursuit Grants, and Math and Science Grants.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


The companion web interface contains numerous features to help users query and navigate the database content. The database content can be freely downloaded and is compatible with third-party Gene Set Enrichment Analysis tools. We expect PAGER 2.0 to become a major resource in integrative GNPA. PAGER 2.0 is available at http://discovery.informatics.uab.edu/PAGER/.Integrative Gene-set, Network and Pathway Analysis (GNPA) is a powerful data analysis approach developed to help interpret high-throughput omics data. In PAGER 1.0, we demonstrated that researchers can gain unbiased and reproducible biological insights with the introduction of PAGs  as the basic data representation elements. In PAGER 2.0, we improve the utility of integrative GNPA by significantly expanding the coverage of PAGs and PAG-to-PAG relationships in the database, defining a new metric to quantify PAG data qualities, and developing new software features to simplify online integrative GNPA. Specifically, we included 84 282 PAGs spanning 24 different data sources that cover human diseases, published gene-expression signatures, drug–gene, miRNA–gene interactions, pathways and tissue-specific gene expressions. We introduced a new normalized Cohesion Coefficient ( Gene-set, Network, and Pathway Analysis’ (GNPA) provides an unbiased approach to analyze the ‘omics’ datasets , first introduced to perform a particular variant of GNPA, has revolutionized the data analysis and interpretation of high-throughput omics data ,5. AccomPAGs—a new unified data structure to represent heterogeneous Pathways (P-type), Annotated-lists (A-type) and Gene-signatures (G-type) (Cohesion Coefficient (CoCo) to assist users in assessing the biological relevance within each PAG. However, there are several limitations, including inadequate PAG coverage, lack of functional information and impact of genes in the PAGs, and lack of molecular interaction/regulation details inside PAGs.To overcome these challenges, we previously developed PAGER, a novel and comprehensive database infrastructure by integrating (G-type) . In PAGEnCoCo to assess the biological relevance of genes inside each PAG, and a new user-friendly interface to help users perform integrative GNPA queries. The new PAGER 2.0 contains 84 282 PAGs, 601 164 gene–gene relationships, and 7 538 275 PAG-to-PAG relationships. The PAGs were derived from 24 different data sources that cover, for example, human diseases, published gene expression signatures, known gene lists affected by shared drugs, pathways, shared miRNA–gene interaction targets, tissue-specifically co-expressed genes and all genes sharing common protein functional annotations. The new normalized CoCo score (nCoCo) employs polynomial regression models to correct for the PAG size bias, which was not considered in the original CoCo score. To assist users in prioritizing genes in the PAGs, we integrated the functional gene–gene interaction data from the recently published HAPPI-2 database (RP-score)   based onhttp://www.proteinlounge.com/Pathway, Pfam . The nuCoCo score , a quality metric to measure PAGs in PAGER 1.0. However, the limitation of the CoCo score is it does not consider the PAG size. Here, we improve the CoCo score to a new PAG size-normalized quality metric. The new score—nCoCo rescales the original CoCo scores based on polynomial regression models to eliminate the PAG size bias . And in the nCoCo score comparison, we were able to explore that quality of PAG WIG001980 is much higher than PAG WIG001424 since the protein–protein interactions (PPIs) of the PAG WIG001980 reached to the upper limit (13 out of 15).We previously developed RP-score gene prioritization algorithm initially reported in  as the web presentation framework and Oracle 12g as the backend database. Real-time calculation of hypergeometric cumulative distribution function (CDF) was implemented with PDL (https://github.com/php-math/PDL), a PHP library for statistics. Cytoscape.js (http://js.cytoscape.org), an open-source graph library, and jQuery were used to visualize gene and PAG networks. D3.js (http://d3js.org/) was used to perform matrix visualizations. DataTables, a plugin for jQuery was used for displaying the tables and enabling download.We used PHP5, Javascript and Codeigniter version 2.1.3 (nCoCo score filtering (nCoCo > 1), the PAGs fold change is 2.86. The m-type PAG-to-PAG relationship fold change is 2.39. The m-type PAG-to-PAG relationship fold change is 1.64. Among the r-type PAG-to-PAG relationship, the sPAG-to-mPAG relationship fold change is 3.96, the mPAG-to-mPAG relationship fold change is 2.13, and the mPAG-to-sPAG relationship fold change is 1.86. This represents a substantial improvement in terms of size and coverage of PAGs and PAG-to-PAG relationships from the previous version.The statistics show that PAGER 2.0 has significantly increased the coverage of the PAGs and PAG-to-PAG relationship in Table To evaluate the statistics of PAGs integrated into PAGER 2.0, we investigated the distribution of the PAG sizes in PAGER 2.0. The peak at the PAG size of 200 in the PAG size distribution shown in Figure nCoCo score for all the PAGs in PAGER 2.0 and compared with PAGER-1.0. The nCoCo score calculation distribution is shown in nCoCo score from PAGER 1.0 and PAGER 2.0 in Figure nCoCo score below the 50% is not significant (0.014 ± 0.010) and similarly not significant when comparing the difference between PAGER 1.0 and PAGER 2.0's nCoCo score above the 50% (0.057 ± 0.032). The break point of the frequency change is due to the inflated nCoCo score = 128 shown in Figure nCoCo score bin of size ranging from 26.8 to 27 in PAGER 2.0 are from DSigDB (48.1 %) and GO term (29.1%). Since the PAGs of DSigDB and GO term consists of functional biological biomarkers, the quality of the PAG is relatively good as the score is concentrated in the bin of size ranging from 26.8 to 27.To evaluate the biological relevance of each PAG in PAGER 2.0, we computed the nCoCo score at the cumulative percentage of 50% (CP50), the quality of the 10 new sources in PAGER 2.0 has been compared with the overall quality of PAGER 2.0 in nCoCo score at CP50 indicates the quality of PAGs from the source. A relative larger nCoCo score at CP50 suggests the distribution of nCoCo is right-skewed. Therefore, the PAGs from DSigDB, Isozyme, GO term and Pfam above the baseline (nCoCo score at CP50 is in the between of 64 and 128) indicate the gene members in the PAGs from these four sources have relatively high interactions and trios. nCoCo score cumulative percentage grouped by type is shown in After calculating the The Users can query the PAGER 2.0 database via the web portal by using the ‘Basic Search’ or ‘Advanced Search’ options. For the ‘Basic Search’, users can query PAGs related to a gene, protein, miRNA, drug or disease. The ‘Basic Search’ results will return a list of PAGs related to the query. For the ‘Advanced Search’ option, users can query a list of genes to retrieve the most similar PAGs in the database.P-value, nCoCo scores, PAG size) tailored to their biological question and context. Finally, we also provide visualization options for the users to explore the PAG-to-PAG relationships in networks or matrix formats ’ (ID = WAG000379) as an example. Figure For every query performed, users can customize the results (columns) of interest in the PAG result page. The results can then be downloaded in common formats, including flat files (comma separated), Excel, and PDF. In addition, we also provide users the option to download the entirety of PAGs in PAGER 2.0 as a GSEA gene set (.gmt) file. The .gmt file format can be directly imported into GSEA to execute the program. Similarly, we allow bulk download of all PAG’s information, PAG-to-PAG relationship and gene–gene relationship contains in PAGER 2.0.The summary of the new features of PAGER 2.0 is in Table nCoCo score is designed for assessing the biological relevance, and gene ranking score (RP-score) is developed to rank the gene member in PAGs, which raises researcher’s interests on network analysis level. The nCoCo score and gene prioritization enable the user to filter the genes in GPNA. In summary, we have updated PAGER 2.0 with new features and data (PAGs coverage and size) that could help users to gain more significant and quantitative biological insights in analyzing their omics datasets. We believe PAGER 2.0 will be a powerful tool and data resource that facilitates the use of GPNA in various omics data and network biology studies.Genes act in concert to drive various biological processes in a complex biological system. High-throughput omics technologies are generating measurements for these biological systems at an unprecedented pace. GNPA provide a powerful approach to analyze and interpret these ‘omics’ datasets to reveal the underlying molecular mechanisms of gene–gene interactions. To facilitate and support GNPA methods, we have developed PAGER 2.0, a comprehensive database that integrates heterogeneous gene-sets, molecular signatures, and pathway/network modules into a unified framework. In PAGER 2.0, we extended the concepts of PAGs and imported new PAGs from 10 sources that increased the amount of PAGs by almost three times. The significant improvement in heterogeneous PAGs definition can assist researchers in acquiring comprehensive insight  of GNPA. The m-type and r-type PAG-to-PAG relationships have been increased by four times. The increased coverage of PAG-to-PAG relationships provides the comprehensive linking between the omics data. The new PAGs' quality measurement, the Supplementary DataClick here for additional data file.


The objective of this research was to assess cropping systems with the potential to produce a reasonable return for farmers while simultaneously reducing the risk of NO3-N movement into these shallow aquifers. From 2009 to 2013 we conducted a field experiment in northwest Iowa in which we evaluated five cropping systems for residual  soil NO3-N content and profitability. Soil samples were taken annually from the top 30 cm of the soil profile in June and August, and from the top 180 cm in November . The November samples were divided into 30 cm increments for analysis. Average residual NO3-N content in the top 180 cm of the soil profile following the 2010 to 2013 cropping years was 134 kg ha-1 for continuous maize (Zea mays L.) with a cereal rye  cover crop, 18 kg ha-1 for perennial grass, 60 kg ha-1 for a three year oat (Avena sativa L.)-alfalfa (Medicago sativa L.)-maize rotation, 85 kg ha-1 for a two year oat/red clover (Trifolium pratense L.)-maize rotation, and 90 kg ha-1 for a three year soybean (Glycine max (L.) Merr.)-winter wheat (Triticum aestivum L.)-maize rotation. However, residual NO3-N in the 90 to 180 cm increment of the soil profile was not significantly higher in the oat-alfalfa-maize cropping system than the perennial grass system. For 2010 to 2013, average profit ($ ha-1 yr-1) was 531 for continuous corn, 347 for soybean-winter wheat-maize, 264 for oat-alfalfa-maize, 140 for oat/red clover-maize, and -384 (loss) for perennial grass. Considering both residual soil NO3-N and profitability data, the oat-alfalfa-maize rotation performed the best in this setting. However, given current economic pressures widespread adoption is likely to require changes in public policy.Many communities in the Midwestern United States obtain their drinking water from shallow alluvial wells that are vulnerable to contamination by NO In solication . As a re0 mg L-1 . In Nebrnce then . Nitratence then .3-N concentrations greater than 10 mg L-1. The city blends water from its shallow wells with water from other sources to ensure the NO3-N concentration in the finished drinking water remains below 10 mg L-1.The city of Sioux Center, located in northwest Iowa, obtains more than 50% of its drinking water from an alluvial aquifer along the West Branch of the Floyd River . The aquifer is accessed by 12 wells that are 9 to 13 m deep. These shallow wells, like those in many Midwestern communities, are strongly influenced by land management practices in the immediate vicinity and in the surrounding watershed. In recent years, several of these wells have yielded water with NO3-N concentrations in water produced by their shallow alluvial wells, can often benefit by altering the vegetation grown in the capture zone surrounding the wells. If the municipality owns the land, it can enroll it in the Conservation Reserve Program–Wellhead Protection Program (CRP-WHP) and receive rental payments for it from the federal government [Midwestern communities like Sioux Center, seeking to reduce NOvernment ,9. Land vernment , indicat3-N losses to groundwater and to be profitable [3-N leaching, while still generating adequate income for the landowner/operator, could facilitate changes in farming practices that improve water quality for many rural communities.Rural communities and private landowners do have other options. Cropping systems utilizing plant species in addition to, or instead of, maize and soybean have the potential to reduce NOofitable –13. The 3-N content, and therefore the risk of NO3-N movement into shallow municipal aquifers. We also wanted to share the results with those who could benefit from the information . We hypothesized that intentionally designed and carefully managed cropping systems would have soil NO3-N contents similar to those in perennial vegetation typical of Conservation Reserve Program plantings, but would generate additional income for the land owner/operator, and therefore be implemented more readily. We successfully evaluated five cropping systems for residual NO3-N content and profitability, and were able to identify several promising crop production strategies for use on land surrounding shallow alluvial wells.The objectives of this research were to assess cropping systems with the potential to: (1) produce a reasonable return for farmers and (2) simultaneously reduce residual soil NO3-N and economic analyses. The site was divided into four blocks (one replicate per block), each containing ten plots. Plots were 18.3 m wide and 190 m long (0.35 ha); large enough to accommodate conventional farm equipment. Crops were harvested from the entire plot area. Soil from each plot was tested annually, and P, K, micronutrients, and lime were applied when necessary to maintain appropriate conditions for crop growth.We conducted our experiment on a 16 ha site 4 km east of Sioux Center, Iowa . The site was about 300 m east of the West Branch of the Floyd River where 12 shallow alluvial wells utilized by the city of Sioux Center were located. The surrounding land area was intensively farmed; the main crops were rainfed maize and soybean. Soils at the site were Mollisols, with the predominant soil type being a Galva silty clay loam . There was no subsurface drainage tile in the site. The entire experimental area had been planted to maize in the previous 3 years, with 2 exceptions. The east 50 m of the experimental area was planted to oats in 2007, and the south half of the experimental area had strips (north-south orientation) of oats, wheat, and maize followed by red clover and cereal rye cover crops planted on it in 2008. To minimize any carry over effects, our plots were oriented at a 90° angle to these strips (east-west orientation) and the data from the 2009 cropping season, although collected and analyzed, was not included in the final soil NOPart of the experimental site was owned by the City of Sioux Center and part was privately owned. The entire site had been farmed by AJS Farms for many years. Permission to conduct the study was obtained from the City of Sioux Center and from Matt and Leon Schuiteman of AJS farms. No protected species were present on the property. No livestock or other animals were involved in the experiment.We evaluated five different cropping systems during 2009 to 2013 growing seasons . DetaileSystem one was continuous maize with a cereal rye cover crop and side-dressed N. This system was included because we wanted to be able to compare well-managed continuous maize with other cropping systems. Plots were strip-tilled  to a depBromus inermis Leyss) and orchard grass (Dactylis glomerata L.) and received minimal N fertilizer . It grew slowly while competing with the oat crop and was not harvested during the establishment year. The following year alfalfa was harvested three or four times, depending on rainfall and subsequent growth. Spring strip tillage, as described for the continuous maize cropping system, was used to prepare the soil for planting maize and to apply starter N fertilizer. Maize was planted in the tilled strips into a living stand of alfalfa. An herbicide application at planting, and again about 4 weeks later, was used to kill the alfalfa and suppress weeds. Maize had excellent tolerance for the herbicide used, and its growth was unaffected by the applications. Nitrogen fertilizer was not side-dressed in this cropping system, except in 2010 when maize was planted in plots that had not had a full year of alfalfa growth.The fourth system was one year of oat under-seeded with red clover followed by one year of maize. Oat and red clover were seeded simultaneously in early spring and the oat crop was harvested for either forage or grain in mid-July or early August, respectively. When the oat crop was harvested red clover plants were still quite small and had little effect on oat grain or forage yield. Red clover was allowed to grow through the fall, overwinter, and continue its growth in the spring. Strip tillage in April was used to prepare 20 cm wide strips for planting and to apply starter N fertilizer. Maize was planted on 76 cm rows into the living red clover cover crop (in the tilled strips). Herbicide was applied at planting and about four weeks later to kill the red clover and suppress weeds. Maize tolerance for the herbicide was excellent, and maize growth appeared to be unaffected by the applications. The late spring nitrate test  was usedThe final system was a three year rotation of soybean, winter wheat followed by a red clover cover crop, and maize followed by a cereal rye cover crop. Strip tillage was used to prepare 20 cm wide strips in early May, and soybeans were planted into the strips in 76 cm wide rows in mid-May. Herbicide applications at planting and about four weeks later were used to kill the cereal rye cover crop and control weedy plant species. Soybeans were harvested in October. N fertilizer was not applied to the soybeans. Winter wheat was planted in early to mid- October following soybean harvest. The winter wheat was harvested for grain in early August, and then a red clover cover crop was planted using a broadcast spreader. Red clover establishment was successful in 2010, but not in other years due to dry soil conditions. Strip tillage the following spring was used to prepare 20 cm wide strips for maize planting and to apply starter N fertilizer. Maize was planted on 76 cm rows into the living red clover cover crop (in the tilled strips). Herbicide was applied at planting and about four weeks later to kill the red clover and suppress weeds. The late spring nitrate test  was used3-N test were completed [3-N test (to a depth of 30 cm) was repeated in late July to early August. Fall maize stalk NO3-N concentrations were determined at the end of the growing season for all maize plots following established protocols [Soil cores (1.9 cm diameter) were taken annually to a depth of 30 cm from each plot in early to mid-June following the protocol for the late spring nitrate test developed by Blackmer . The soiompleted . The soirotocols . Stalk s3-N content. For every plot and 30 cm soil increment, the 18 soil cores were composited. Composited samples from all plots and soil depth increments were refrigerated before shipment to Midwest Labs, where they were dried and analyzed. Midwest Labs determined the NO3-N concentration in each soil sample and reported it in mg kg-1. In November of 2016 we took 20 soil cores 4 cm in diameter and 180 cm deep from the east 2/3 of the plot area, and another 20 samples from the west 1/3 of the plot area. Samples were divided into 30 cm increments, composited, and shipped to Midwest Labs for bulk density analysis. Results from both plot areas were very similar. Mean bulk density for each soil depth  was used to calculate soil NO3-N content (kg ha-1). The resulting data was used to construct a NO3-N content profile (180 cm deep in 30 cm increments) for the soil in each plot and to document the impact of crop rotations and weather on the profile over time [In late October and November of each year, 18 soil cores 4 cm in diameter were collected from each plot with a hydraulic probe. Samples were taken to a depth of 180 cm and divided into 30 cm increments for analysis of residual (at the end of the growing season) NO-1 for maize, 130 g kg-1 for soybean, 140 g kg-1 for oat, 135 g kg-1 for wheat grain, 100 g kg-1 for straw, and 150 g kg-1 for alfalfa and perennial grass. P and K removal rates were calculated for each crop based on actual yields, and an economic cost for nutrient replacement was assessed to each crop in each year. An opportunity cost was charged for money that was tied up in variable expenses related to the enterprise. Costs for field operations such as fertilizer applications, planting, tillage, and harvesting were assigned based on the Iowa Farm Custom Rate Survey [-1 yr-1 for 2010–2012. Due to farm program changes there was no direct payment for 2013. Profitability (economic profit) for each cropping system was determined by subtracting expenses from total revenue.An enterprise analysis was conducted for each cropping system for the 2010 to 2013 cropping seasons. Actual costs were used for seed, herbicides, N fertilizer and other direct inputs. Yields of grain and forage were adjusted for moisture content. We used a standard moisture content of 150 g kge Survey . These rA randomized complete block design with four replications was used for the experiment. Each crop phase of each rotation system was present every year so we could accurately assess cropping system effects . Each ye3-N concentration (Mg m-3) was analyzed using the procedures for a split-split-plot design with years as main plots, cropping systems as sub-plots, and position in the soil profile (soil depth increments) as sub-sub-plots. Repeated measures of NO3-N content within a cropping year were analyzed using the nlme, lme4 and lmerTest packages in the R Project for Statistical Computing [An analysis of variance was conducted for the data from each year using Statistix . Multiplomputing  software3-N content in the upper 180 cm of the soil profile  NO profile . Continucrements , even ther crops –28 and ser crops  can reduer crops ,30. The leaching . In the al soils . It is aal soils . In addial soils . The oatrotation ,30. In ahe maize . The soytrations . Winter 3-N concentration from the intermediate depth increment (60 to 90 cm) to the deepest depth increment (150 to 180 cm), while those that included only annual crops  showed clear increases  did not show an increase in mean NOncreases . Our rest 150 to 0 cm, whinditions ,35.-1 of NO3-N in the 0 to 180 cm increment of the soil profile, while the perennial grass system had only 18 kg ha-1  than the oat/red clover-maize (85 kg ha-1) or the soybean-winter wheat-maize/cereal rye (90 kg ha-1).On average, the continuous maize/cereal rye system had a total of 134 kg ha kg ha-1 . The oth3-N contents in the 0 to 90 cm increment of the soil profile were higher than in the 90 to 180 cm increment for all cropping systems (3-N content in the 0 to 90 cm increment of the soil profile (88.8 kg ha-1) than the other cropping systems. Perennial grass had the lowest residual NO3-N content in the 0 to 90 cm increment of the soil profile (10.6 kg ha-1), followed by the oat-alfalfa-maize system (48.7 kg ha-1). Residual NO3-N content in the 0 to 90 cm increment was similar for oat/red clover-maize (69.5 kg ha-1) and soybean-winter wheat-maize/cereal rye (65.1 kg ha-1) systems.Mean NO systems , S2 File3-N throughout the entire soil profile is potentially mobile, the NO3-N in the deeper portion is closer to the water table and more difficult for plant roots to access, making it more likely to leach into groundwater and/or into subsurface drainage tile. The continuous maize system had the highest residual NO3-N content (45.2 kg ha-1) in the 90 to 180 cm increment of the soil profile, while the perennial grass system had the lowest (7.2 kg ha-1). However, the residual NO3-N content for the oat-alfalfa-maize system between 90 and 180 cm (11.6 kg ha-1) was not significantly higher than for perennial grass. The oat/red clover-maize system had a significantly higher NO3-N content (15.0 kg ha-1) than perennial grass in the 90 to 180 cm depth increment, but was not different than the oat-alfalfa-maize system. The NO3-N content in the 90 to 180 cm increment for the soybean-winter wheat-maize system (29.5 kg ha-1) was significantly lower than for maize, but higher than for the other cropping systems. This may have been due in part to the inclusion of soybean in this system, as other researchers have suggested that soybean may contribute to NO3-N export from agricultural watersheds [While NOtersheds .3-N content in the top 30 cm of soil throughout the growing season provides additional insights into the dynamics of NO3-N behavior . This rate was based on the late spring nitrate test [-1 recommended by Iowa State University for continuous maize [3-N concentrations, indicating that the maize plants had adequate N availability during the growing season, are between 700 and 2000 ppm. The highest average stalk nitrate value we observed was 1220 ppm, and the means for the maize crops were between 441 and 482 ppm. This data indicates that N availability, and our N fertilizer rates, were on the conservative (low) end of the spectrum.The comparatively high NOate test  and is sus maize . The falus maize  was used3-N content was heavily influenced by precipitation. The 2011 and 2012 cropping seasons were both unusually dry  and lowest for the perennial grass cropping system ($916 ha-1). Average expenses for the other three cropping systems were similar ($1250 to $1342 ha-1)  .Yields of soybean and oat were fairly stable during the 2010 to 2013 cropping seasons, while those of maize, perennial grass, alfalfa and winter wheat were more variable . Dry con-1 in 2012, but only $171 Mg-1 in 2013. Perennial grass hay was valued at $88 Mg-1 in 2010, and increased to $198 Mg-1 in 2012. Oat and winter wheat straw were valued at $77 Mg-1 in 2010 and $132 Mg-1 in 2011.Market prices varied greatly from one year to the next . For exa-1) and perennial grass was the least profitable ($-384 ha-1). However, perennial grass was primarily grown as a control treatment and therefore did not receive N fertilizer. Appropriate fertilization would likely have increased yields with little or no increase in soil nitrate concentrations [-1, the oat/red clover-maize system $140 ha-1 and the soybean-winter wheat-maize/cereal rye $347 ha-1.Profitability of each cropping system was estimated using three different procedures . This watrations ,18,39. T3-N and moderate to high profitability don’t seem to occur simultaneously. The cropping system with the lowest residual soil NO3-N levels  was the least profitable, and the system with the highest soil NO3-N levels  was the most profitable  for seed production [3-N concentrations compared to annual crops [Currently, the crops that most effectively reduce residual soil NOoduction . This neal crops . Product3-N content [Breeding maize and other annual crops for increased ability to extract soil N at low concentrations may also be feasible –48. Rece content .3-N leaching from crop fields, agricultural policy needs to reward management practices that re-couple the C and N cycles, and prevent nitrate from moving down through the soil profile [3-N concentrations in the lower levels of the soil profile. Alternatively, eligibility for federal government farm program benefits in sensitive areas could be dependent on including such a crop in rotation. The removal of nitrogen from the lower soil profile in wellhead capture zones, and on the broader landscape, represents a common good for society and therefore should be promoted by a range of well-designed and effective public policies.To successfully reduce NO profile . The USD profile , as is tS1 File(DOCX)Click here for additional data file.S2 FileData points represent means of the 2010 to 2013 cropping years.(DOCX)Click here for additional data file.S3 File(JPG)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(DOCX)Click here for additional data file.S7 File(DOCX)Click here for additional data file.S8 File(DOCX)Click here for additional data file.S9 File(DOCX)Click here for additional data file.


Overall, these results suggest a potential pharmaceutical feature of CA in the treatment of BPH.Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in male population, of which incidence increases gradually with age. In this study, we investigated the effect of chrysophanic acid (CA) on BPH. BPH was induced by a 4-week injection of testosterone propionate (TP). Four weeks of further injection with vehicle, TP, TP + CA, TP + finasteride was carried on. In the CA treatment group, the prostate weight was reduced and the TP-induced histological changes were restored as the normal control group. CA treatment suppressed the TP-elevated prostate specific antigen (PSA) expression. In addition, 5α-reductase, a crucial factor in BPH development, was suppressed to the normal level close to the control group by CA treatment. The elevated expressions of androgen receptor (AR), estrogen receptor α and steroid receptor coactivator 1 by TP administration were also inhibited in the CA group when compared to the TP-induced BPH group. Then we evaluated the changes in three major factors of the mitogen-activated protein kinase chain during prostatic hyperplasia; extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). While ERK was elevated in the process of BPH, JNK and p38 was not changed. This up-regulated ERK was also reduced as normal by CA treatment. Further  The term BPH stands for the histological diagnosis of unregulated proliferation in the prostate tissue, however, the recent main focus of BPH moves from the justification of BPH on to the major symptoms of it, which can negatively impact the quality of the patient's life. The clinical symptoms of BPH are urgency, frequency, weak stream, nocturia and incomplete emptying of urine, which are also known as lower urinary tract symptoms (LUTS) . Casabe  disease .Although the mechanism of prostatic enlargement is not totally clear, the relevance of androgens is considered to be one of the most important factors. Androgens may not be the direct cause of BPH, but still the testicular androgens are essential in the development of prostatic growth . Among t1-adrenergic receptors, is a common choice for initial therapy in the USA and Europe [Serenoa repens [BPH patients suffering from severe symptoms need to be treated with surgery or medication. Although transurethral resection of the prostate is the most common surgical treatment for BPH, this procedure can lead to negative complications, for instance bleeding, urethral stricture, and incontinence . The mosd Europe –17. Theyd Europe . On the d Europe . But 5ARd Europe . Therefod Europe . USA anda repens .Chrysophanic acid (CA) is a member of the anthraquinone family. Previous studies have shown that the derivatives of anthraquinones exert a number of biological effects including anticancer , 24, hepet al. have reported increased prostate weight by treatment with testosterone propionate (TP) in 1965 [Since Kato  in 1965 , the rat in 1965 , current in 1965 , severalIn this study, we demonstrated the effects of CA on BPH in TP-induced BPH rats by measuring the prostate tissue weight, examining the histological changes, and evaluating the major factors involved in the biology of BPH such as prostate specific antigen (PSA), 5AR, AR, SRC1, estrogen receptor α (ERα), and mitogen-activated protein kinases (MAPKs).The body weights, prostate weights, and the prostate indexes of the rats are shown in Table Figure The prostate weight index was calculated dividing total prostate tissue weight (mg) by body weight (100 g). As shown in Figure To evaluate the histological changes in the prostates, an H&E staining analysis was conducted. As in Figure Figure p < 0.05) in the prostatic lumen areas when compared with the BPH group. Daily treatment of Fi for 4 weeks  assay and a western blot analysis was carried on. Figure To confirm the former results in the protein level, a western blot assay was performed. As in Figure AR, one of the major key factors in development of BPH, was evaluated by microscopic examination of the immunostained prostate slides Figure . The TP-SRC1 interacts with AR and enhances both ligand-dependent and independent transactivation to increase transcription of androgen-regulated genes , 13. TheSubstantial evidence has shown that stimulation of ERα and ERβ in the prostate can drive either proliferation or anti-proliferative mechanisms, respectively . The immTo confirm the effect of CA on ERα in the protein level, a western blot assay was carried on. As shown in Figure The MAPK pathway is a protein chain which is closely related to cell proliferation and apoptosis . Howeverin vitro studies. An MTS assay was conducted in order to assess possible cytotoxicity of CA in RWPE-1 cells. As a result, CA did not affect the viability of RWPE-1 cells up to the concentration of 5 μM (data not shown). Next, we have established a BPH-like in vitro model by treating RWPE-1 cells with 0.5 μM of TP, a model which was modified from the study by Ren et al. [vivo study, and 5 μM of CA successfully inhibited this increase of p-ERK  in a form of brownish-yellow powder. It is known to be abundant in the Traditional Korean Medicinal herb Rheum palmatum L., which is frequently prescribed for constipation. Numerous reports of CA are published, but to date, there are no reports on the effects of CA in BPH. Therefore, in this study, we investigated the effects of CA using the TP-induced BPH rat model, in order to estimate the new therapeutic potential of CA in BPH treatment. As a result, CA treatment successfully reduced the enlarged prostate tissue by TP administration  was purchased from Sigma Chemicals  and was dissolved in 100% dimethyl sulfoxide (DMSO). TP was provided from Wako pure chemical industries , and Fi (≥ 97 % pure) was purchased from Sigma-Aldrich Inc. . Antibodies for AR, ERα and SRC1 were purchased from Pierce biotechnology , antibody for 5AR was a product from Abcam Inc. . The Antibodies for PSA, p38, and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology . p-p38, p-ERK, p-JNK, and JNK were purchased from Cell Signaling Technology . Anti-ERK antibody was obtained from Bioworld Technology .n = 28) with initial body weights of 200-220g were purchased from the Dae-Han Experimental Animal Center . The animals were all maintained in conditions in accordance with the regulation issued by the Institutional Review Board of Kyung Hee University (confirmation number: KHUASP(SE)-P-034). The rats were housed in a pathogen-free room maintained at 23 ± 2°C and a relative humidity of 70% with an alternating 12 h light/dark cycle. Water and standard laboratory diet  were provided ad libitum.12-week-old male Sprague-Dawley (SD) rats (n = 28) as described previously [2. Blood samples were obtained from the caudal vena cava. The blood containing tubes were remained at RT for 2 h and sera were separated by centrifuging at 3000 × g for 20 min in 4°C. The serum was stored at -80°C until further assays. The intact prostate tissue was carefully dissociated and removed, washed with PBS, and then weighed. Relative prostate weight was calculated as the ratio of prostate weight (mg) to body weight (100 g). The prostate tissue was divided in half; one half was fixed in 10% formalin and embedded in paraffin for histomorphological assays, the other was stored at -80°C for further assays.BPH was induced by a pre-4-week treatment of daily subcutaneous injections of TP (5 mg/kg) at the inguinal region . RWPE-1 cells were cultured in Roswell Park Memorial Institute medium (RPMI)  supplemented with 100 mg/ml penicillin/streptomycin  and 10% FBS (Sigma-Aldrich Inc.). After 24 h of incubation, the culture media was replaced with fresh media containing 0.5 μM of TP in order to induce cell proliferation. CA was supplemented together within TP-containing media.4 cell/well) and incubated in RPMI plus 10% FBS for 24 h. Then the cells were incubated in fresh media containing various concentrations of CA for an additional 24 h. Cell viability was monitored using the cell proliferation MTS kit by the Promega Corporation as recommended by the manufacturer. Prior to measuring the viability, the media were removed and replaced with 200 μl of fresh RPMI plus 10% FBS medium and 10 μl of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution. The cells were then incubated in the incubator for 4 h. The absorbance was measured at 490 nm in a VERSAmax microplate reader  to determine the formazan concentration, which is proportional to the number of live cells.RWPE-1 cells were seeded  according to the manufacturer´s protocol. Briefly, cells were plated overnight and labeled with 10 μM EdU for 24 h, and then the cells were transferred into a 6-well plate and fixed with 3.7% formaldehyde for 15 min at RT, washed twice with 3% BSA in PBS. Next, 1 ml of 0.5% Triton X-100 in PBS was added, followed by a 20 min incubation at RT. EdU was detected by staining cells with Click-iT reaction cocktail for 30 min at RT. DNA staining was performed using Alexa Fluor 488 Imaging Kit . Samples were analyzed using iRiS Digital Cell Imaging System  at 488 nm.t-test using IBM SPSS Statistics 22 software . Values with P < 0.05 were considered as statistical significance.The data values were presented as the mean ± standard deviation (S.D.). Differences in mean values were analyzed by one-tailed Student's


Among the 36 patients participating in this study, a subgroup of 9 patients developed liver stiffness >6.3 kPa after 4–5 years with an increase of ΔTE >0.38 kPa/a (the group with increasing liver stiffness was labelled TEinc). APRI- and FIB-4 scores confirmed the rationale for grouping. The frequency of CFLD assessed by conventional diagnosis was significantly higher in TEinc-group compared to the control group (TEnorm). None of the adult CF patients matched criteria for TEinc-group. For the first time it was shown that the non-invasive longitudinal assessment of TE allows identification of patients with progression of CFLD in a subgroup of juvenile but not in adult CF patients. Comparing TE to conventional fibrosis-scores underlined the strength of the continuous assessment of liver stiffness for the exact diagnosis of progressive CFLD. The newly described cut-off for pathologic increase of liver stiffness, ΔTEcutoff = 0.38kPa/a, might enable to detect developing CFLD using consequent follow up TE measurements before reaching the level of stiffness indicating established CFLD. Nevertheless, the limited size of the analyzed cohort should encourage a prospective, multi-center, long term follow up study to confirm the suggested cut-off for the rise in liver stiffness.About 30% of patients with Cystic Fibrosis (CF) develop CF-associated liver disease (CFLD). Recent studies have shown that transient elastography (TE), as a method to quantify liver stiffness, allows non-invasive diagnosis of CFLD in adults and children with CF. Within this study we aimed to prospectively identify patients at risk for development of CFLD by longitudinal analysis of liver stiffness and fibrosis scores in a 5-year follow-up. 36 pediatric and 16 adult patients with initial liver stiffness below the cut-off value indicative of CFLD (6.3 kPa) were examined by transient elastography for 4–5 years. TE, APRI-, and FIB-4-scores were assessed and compared by Kruskal-Wallis test and receiver operating characteristic (ROC)-analysis. Frequencies were compared by Chi Cystic fibrosis (CF) is the most common multisystem autosomal recessive genetic disorder with a high morbidity and mortality in Caucasian population with an incidence of approximately 1 of 3500 births worldwide . HoweverThis prospective 5-years follow-up study was performed during the period of 2009–2015. It has been conducted according to the principles of the Declaration of Helsinki and approved by the ethics committee of the medical faculty of the Justus-Liebig-University Giessen  with the approval No. 75/09.All participating adult patients and the parents of the pediatric patients received, signed and agreed with the written informed consent for the procedure for the upcoming period of minimum five years in all cases. The treatment meets all requirements of the European and U.S. guidelines .The diagnosis of CF was an essential prerequisite for participating in this study and was established by the sweat chloride test with application of pilocarpine and the positive gene mutation tests with elevated immunoreactive trypsinogen (IRT) in all subjects.In total, 173 out-patient positive cystic fibrosis (CF) patients, 85 males and 88 females, with a median age of 13 (2–47), were treated at the Department of Internal Medicine at the University Hospital Giessen. The cohort was classified into patients with liver disease (CFLD) on the one hand or without liver disease (CFnoLD) on the other hand according to the clinical guideline criteria published by Debray in 2011 [hepatomegaly with or without a splenomegalytwo abnormal serum liver enzyme levels other ultrasound parameters of liver abnormalities than hepatomegaly such as varied echogenicity, increased density, nodules, rough liver margins, evidence for portal hypertension Patients with other diagnosed causes for chronic hepatobiliary diseases were excluded of this study .All patients underwent clinical examination at intervals of approximately once a year.In addition, each patient received regular haematological and biochemical investigation as well as a pulmonary function test  ,10.APRI  and FIB-4 (Fibrosis-4 = Age (years) x AST (U/l) / Platelets 109/L x 3 ALT (U/l)) which are validated to detect fibrosis and predict cystic fibrosis liver disease (CFLD), as described by Leung et al. [Results were computed to g et al. .Furthermore, all patients underwent a regular abdominal ultrasound examination (Philips HD 11X E) as well as an examination with the transient elastography (TE) with FibroScan®.Our study focuses on juvenile patients, whose valid and complete measures were conducted over 4–5 years. Those with an already existing high liver stiffness (above the cut-off) were excluded from this study.Assessment of liver stiffness was performed as described before .After collecting the patients´ demographic and clinical characteristics of up to 5 years, IBM SPSS Statistics 22.0  was used to summarize and perform the statistical analysis.Differences in the TE values, ΔTE, median TE (0–5), as well as APRI- and FIB-4-score were assessed and compared in multiple tests using the Kruskal-Wallis-test.The values of the liver stiffness measured with TE, APRI- and FIB-4-score are visualized in Box- and- Whisker-Plots. Outliers (o) define the values deviating from the box at 1.5- to 3-fold interquartile range. Significant and highly significant differences are pointed out . Pearson’s Chi-square test compares and differentiates between the frequency of CFnoLD and CFLD in the two subgroups.The diagnostic performance and accuracy of TE, APRI, FIB-4 and ALT were assessed by using the receiver operating characteristics (ROC)-analysis.p<0.001, ALT(CFnoLD) = 24.7±1.6 U/l and ALT(CFLD) = 39.2±4.7 U/l with p<0.003, APRI(CFnoLD) = 0.20±0.02 and APRI(CFLD) = 0.36±0.08 with p = 0.007. The values were calculated as means of all measurements at all time points during the follow-up. In theROC-analysis, the following diagnostic accuracies of TE, serum ALT, APRI- and FIB-4 scores for the distinction between CFLD and CFnoLD were calculated: AUC(TE) = 0.91±0.05 with p<0.001, AUC(ALT) = 0.76±0.08 with p = 0.003, AUC(APRI) = 0.74±0.08 with p = 0.007, AUC(FIB-4) = 0.62±0.09 with p = 0.17. These data are consistent with the results of previous studies [For 60 of the 173 CF patients, including 16 adults and 44 children, follow-up data with initial and regularly consecutive TE over 4–5 years and quantification of APRI- and FIB-4 scores were available . 21 of tThis prospective identification of pediatric patients at risk for the development of CFLD, analyzing the liver stiffness and the fibrosis scores longitudinally, required the following stratification : 36 chilcut-off = 0.38kPa/year (inc with ΔTEinc>0.38kPa/year). 27 pediatric CF patients (75%) exhibited a stable stiffness value below the threshold of 0.38 kPa/year. These patients were labelled TEcon and served as a control group for those 9 (25%) pediatric CF patients with a continuously increasing liver stiffness (target group TEinc). Fibrosis scores APRI and FIB-4 as well as clinical parameters specific for liver disease were assessed.10 (28%) out of 36 pediatric CF patients had a final stiffness above the threshold of 6.3 kPa in the fourth and fifth year. In order to identify those patients with a continuous rise of liver stiffness and final stiffness above the threshold of 6.3 kPa, the optimal cut-off for the mean increase in TE was calculated by ROC analysis to be ΔTEkPa/year . Eventuainc and TEcon did not differ in the baseline demographics and clinical data such as age, BMI, or the involvement of the pancreas and liver enzyme levels. Furthermore, there was no difference in the liver stiffness and fibrosis scores at the beginning of the study between the patients with stable liver stiffness over time and those with exacerbation.As shown in con and TEinc patients were stratified according to the criteria for CFLD diagnosis by Debray et al [2 = 0.001(**). As illustrated in con patients only 5 (18.5%) patients developed CFLD during the follow-up period, as diagnosed by guideline criteria. In contrast, out of the 9 patients within the TEinc group, 8 patients (= 88.9%) were diagnosed with CFLD according to the criteria by Debray during the follow-up. con and TEinc. The median values of the yearly increase of liver stiffness were ΔTEinc = 0.82 kPa (0.46–1.79) and ΔTEcon = -0.05 kPa (-0.76–0.3) with p<0.001. Similarly, the difference in median stiffness definitely supported the distinction of the two groups: median TEcon(year 0–5) = 4.5 kPa and median TEinc(year 0–5) = 5.45 kPa, p<0.001  in the fourth and fifth year of the follow-up (p = 0.003) at the fourth year of the follow-up while the APRI score increased tendentially but did not reach the level of significance .In the TEollow-up . This inificance . Both, ACFLD is a relatively frequent and an early complication of CF develops in 5–10% of children within their first decade of life. There is also a chance of 4.5% of an incidence of cirrhosis during a median period of 5 years from the time of the diagnosis of the liver disease .Given these considerations, the early diagnosis of CFLD is urgently needed, and patients with an early stage liver disease are more likely to benefit from therapy with ursodeoxycholic acid (UDCA) . AlthougNevertheless, so far only two studies presented follow-up data for TE-assessment in CFLD patients ,12. Karlinc-group were also identified to have CFLD ofy comprehensive conventional criteria [inc group. According to previously published data [8 out of 9 children of the TEhed data , no incrhed data .A recently published, liver biopsy based study showed that the biomarker scores APRI and FIB-4 differentiate between CFLD and CFnoLD in pediatric CFLD patients . TherefoA possible limitation of our study might be the lack of histopathologic assessment of CFLD in our cohort. However, due to the focal nature of CFLD, liver biopsy is controversially discussed in CFLD and thus not acting as regular diagnostic ,16. MoreThis study can be distinguished from other studies, as it is a prospective long term observation of CF patients to be at risk for CFLD. Furthermore we compared TE with established histology proven fibrosis scores (APRI and FIB-4). Our newly described cut-off for pathologic increase of liver stiffness might enable to detect developing CFLD using consequent follow up TE measurements before reaching the level of stiffness indicating established CFLD. With respect to the limited size of the analyzed cohort, we perceive our study as a signal to encourage a prospective, multi-center, long term follow up study to confirm our data and to refine the suggested cut-off for the rise in liver stiffness for the identification of children at risk for development of CFLD.